A fundamental question in cell biology is how the recruitment of proteins to cellular membranes is achieved and regulated. The long term objective of the proposed research is to characterize the structural and energetic basis for the binding of peripheral membrane proteins to phospholipid membranes and, in turn, to better understand the biophysical basis of membrane-mediated events in cells. The overall strategy is based on two distinct computational approaches: 1) the calculation of the physical interactions between proteins and membranes, and 2) bioinformatics tools for sequence analysis, structure comparison and structure prediction. By studying different membrane-interacting proteins in parallel, two hypotheses will be tested: 1) that two physical factors-electrostatics and hydrophobicity-are the major determinants of membrane binding, and 2) that these physical factors are manifested as patterns in sequence, structure and biophysical characteristics that can be used to predict membrane targeting potential. The first specific aim is to describe how nonspecific electrostatic and hydrophobic interactions mediate the wide range of membrane binding behaviors exhibited by secreted phospholipases A2. The second specific aim is to understand the role of electrostatic interactions in the calcium-dependent and independent membrane binding of C2 domains. The third specific aim is to develop structural models for phosphoinositides, an important class of signaling lipids. The fourth specific aim is to determine the energetic basis of both the specific and non-specific interactions of pleckstrin homology (PH) domains with membranes containing phosphoinositides. The computational results will be used in the design and interpretation of experiments through collaborations with experimental groups and will lead to rules that can be used to detect membrane targeting motifs in proteins. Secreted phospholipases A2 have been implicated in inflammation, and C2 and PH domain-containing proteins involved in phosphoinositide signaling have been implicated in oncogenesis; these proteins require membrane association for their function. Thus, a detailed understanding of the molecular mechanisms underlying the control of membrane association would facilitate the rational design of drugs that inhibit membrane binding.